Breast cancer (BC) is the most common malignancy among women worldwide. Around 15–25% of BC overexpress the human epidermal growth factor receptor 2 (HER2), which is associated with a worse prognosis and shortened disease-free survival. Therefore, anti-HER2 therapies have been developed, such as monoclonal antibodies (trastuzumab, Tz), antibody–drug conjugates (ado-trastuzumab emtansine, T-DM1), and pharmacological inhibitors of tyrosine kinase activity (lapatinib, Lp). Although Tz, the standard treatment, has significantly improved the prognosis of patients, resistance still affects a significant population of women and is currently a major challenge in clinical oncology. Therefore, this study aims to identify potential biomarkers to predict disease progression (prognostic markers) and the efficacy of Tz treatment (predictive markers) in patients with HER2+ BC. We hypothesize that proteins involved in cell motility are implicated in Tz-resistance. We aim to identify alterations in Tz-resistant cells to guide more efficient oncologic decisions. By bioinformatics, we selected candidate proteins and determined how their expression, localization, and the process they modulate were affected by anti-HER2 treatments. Next, using HER2+ BC patients’ data, we assessed these proteins as prognostic and predictive biomarkers. Finally, using Tz-resistant cells, we evaluated their roles in Tz response. We identified deregulated genes associated with cell motility in Tz/T-DM1-resistant vs. -sensitive cells. We showed that Tz, T-DM1, and Lp decrease cell viability, and their effect is enhanced in combinations. We determined synergism between Tz/T-DM1 and Lp, making possible a dose reduction of each drug to achieve the same therapeutic effect. We found that combinations (Tz/T-DM1 + Lp) efficiently inhibit cell adhesion and migration. Furthermore, we demonstrated the induction of FAK nuclear and cortactin peri-nuclear localization after T-DM1, Lp, and Tz/T-DM1 + Lp treatments. In parallel, we observed that combined treatments downregulate proteins essential for metastatic dissemination, such as SRC, FAK, and paxillin. We found that low vinculin (VCL) and cortactin (CTTN) mRNA expression predicts favorable survival rates and has diagnostic value to discriminate between Tz-sensible and Tz-resistant HER2+ BC patients. Finally, we confirmed that vinculin and cortactin are overexpressed in Tz-resistance cells, SKBR3-RTz. Moreover, we found that Tz plus FAK/paxillin/cortactin-silencing reduced cell adhesion/migration capacity in Tz-sensitive and -resistant cells. In conclusion, we demonstrate that combined therapies are encouraging since low doses of Tz/T-DM1 + Lp inhibit metastatic processes by downregulating critical protein expression and affecting its subcellular localization. We propose that vinculin and cortactin might contribute to Tz-sensibility/resistance in BC cells. Finally, we identify potential prognostic and predictive biomarkers that are promising for personalized BC management that would allow efficient patient selection in order to mitigate resistance and maximize the safety and efficacy of anti-HER2 therapies.
乳腺癌是全球女性最常见的恶性肿瘤。约15-25%的乳腺癌存在人表皮生长因子受体2（HER2）过表达，这与不良预后及较短的无病生存期相关。因此，针对HER2的靶向治疗药物相继问世，包括单克隆抗体（曲妥珠单抗，Tz）、抗体偶联药物（恩美曲妥珠单抗，T-DM1）以及酪氨酸激酶活性抑制剂（拉帕替尼，Lp）。尽管标准治疗药物Tz已显著改善患者预后，但耐药问题仍影响大量女性患者，成为当前临床肿瘤学面临的主要挑战。本研究旨在探索可用于预测HER2阳性乳腺癌疾病进展（预后标志物）及Tz治疗疗效（预测标志物）的潜在生物标志物。我们提出假设：参与细胞运动的相关蛋白与Tz耐药机制存在关联，并试图通过识别Tz耐药细胞的生物学改变来指导更有效的肿瘤治疗决策。 通过生物信息学分析，我们筛选出候选蛋白，并检测了抗HER2治疗对其表达水平、亚细胞定位及调控过程的影响。随后，基于HER2阳性乳腺癌患者数据，评估了这些蛋白作为预后及预测性生物标志物的价值。最后，利用Tz耐药细胞模型，验证了其在Tz治疗反应中的作用机制。 研究发现，相较于敏感细胞，Tz/T-DM1耐药细胞中存在与细胞运动相关的基因表达失调。实验证实Tz、T-DM1和Lp均能降低细胞活力，且联合用药可增强此效应。我们确定了Tz/T-DM1与Lp的协同作用，使得在达到相同疗效时可降低各药物剂量。联合方案（Tz/T-DM1 + Lp）能有效抑制细胞黏附与迁移。此外，研究显示T-DM1、Lp及Tz/T-DM1 + Lp处理可诱导黏着斑激酶（FAK）核转位及皮层蛋白（cortactin）核周聚集。同时观察到联合治疗能下调SRC、FAK和桩蛋白（paxillin）等转移扩散关键蛋白的表达。 临床数据分析发现，低表达的纽蛋白（VCL）与皮层蛋白（CTTN）mRNA可预测良好生存率，并具有鉴别Tz敏感与耐药HER2阳性乳腺癌患者的诊断价值。实验进一步证实，在Tz耐药细胞SKBR3-RTz中VCL与CTTN存在过表达。此外，Tz联合FAK/桩蛋白/皮层蛋白基因沉默能降低Tz敏感与耐药细胞的黏附/迁移能力。 综上所述，本研究证实联合治疗方案具有应用前景，低剂量Tz/T-DM1 + Lp可通过下调关键蛋白表达并影响其亚细胞定位来抑制转移进程。我们提出VCL与CTTN可能参与调控乳腺癌细胞对Tz的敏感性/耐药性。最终，本研究鉴定出具有潜力的预后与预测性生物标志物，为乳腺癌个体化治疗提供新方向，有助于通过精准患者筛选来克服耐药问题，最大化抗HER2治疗的安全性与有效性。
Potential Biomarkers Associated with Prognosis and Trastuzumab Response in HER2+ Breast Cancer
肿瘤(癌症)患者之家